Nano-liquid chromatography mass spectrometry (nanoLC/MS) has emerged as the gold standard for proteomic and glycomic laboratories. The combined nanoLC/MS technique is capable of resolving highly complex mixtures. The mixtures having components covering a wide dynamic range. The small bore LC/MS device obtains valuable mass spectral data, and is ultimately capable of identifying the components in the mixture. Furthermore, nanoLC/MS identifies, localizes, and structurally characterizes the subtle chemical variations between sample components, such as post-translational modifications.
Quantitative proteomic profiling using nanoLC/MS is an emerging technology having a great potential for the functional analysis of biological systems and for the detection of clinical diagnostic marker proteins. Quantitative proteomic profiling has been demonstrated for quantitation of proteins, as well as specifically for phosphoproteins and glycoproteins. In addition to protein identification, characterization of post-translational modifications, and quantitation of protein differential expression, nanoLC/MS is also useful for the investigation of protein-protein complexes. NanoLC/MS is a far-reaching technology, positively impacting many areas of proteomics, and consequently is invaluable to biological laboratories.
NanoLC was developed in the 1980's and has become an accepted, indispensable tool for resolving highly complex, otherwise intractable biological mixtures. NanoLC only requires attomole to femtomole sample amounts and offers high sensitivity because of the resolving power. High LC separation efficiency has resulted in 3-fold increases in the number of peptides detected by electrospray Fourier transform ion cyclotron resonance MS for proteomic analyses. Furthermore, high-efficiency separations result in narrower, sharper peaks which enables more sensitive MS detection for low abundance peptides. As a result, more complex problems are now addressable such as molecular interactions, ion structures, quantitation, and kinetics in the both the field of proteomics and glycomics.
Conventional nanoLC uses chromatography columns having inner diameters ranging from 25-150 μm. The columns are packed with 1-10 μm stationary phase particles. The most typical column has a 75 μm inner diameter and is packed with 1-5 μm particles. Typical nanoLC flow rates range from 50-700 nL/min. Smaller particle sizes and longer columns generate higher resolving power, but also increased backpressure on the system. Other low flow separation techniques including capillary electrophoresis, capillary zone electrophoresis, and capillary electrochromatography offer high sensitivity, but are difficult to couple to mass spectrometry. Furthermore, these alternative techniques have limited sample loading volumes.
Small bore LC columns are typically made by first placing a frit in a capillary and then packing the capillary with a sorbent material. The porous frits have been made from porous filters, scintering methods, restrictors and tapers including nanoelectrospray emitters, porous ceramic plugs, sol-gel technology, unions containing stainless steel screens, and self-assembled particles. Columns can be packed by a variety of different methods including dry packing, supercritical packing, electrokinetic packing, sol-gel packing, centripetal force packing, or the most widely accepted, slurry packing. A great number of variables encountered in the manufacturing of the columns affect the separation efficiency of the final product. These variables include the sorbent material itself, the type of solvent used in the packing slurry, the slurry concentration, packing pressure, and the frit type. Consequently, it is widely accepted that small bore LC column manufacturing is very difficult and can even be considered an art form. One particular problem is that voids in the packed bed that are formed within the column during manufacturing have a detrimental effect on column performance and efficiency. Therefore, the manufacturing of these small bore LC columns is challenging. Although high level research groups are able to fabricate their own small bore LC columns, the average nanoLC user must purchase columns from a commercial source.
It is evident that much of the actual manufacturing process for small bore LC columns is performed “by hand” in a non-automated fashion. Because of this, every column made by a reputable vendor undergoes rigorous and time consuming quality control testing which includes performing an analysis on each column prior to shipment. This additional testing drives up the cost of commercially available small bore LC columns. The materials used to make a small bore LC column are inexpensive. However, the manufacturing costs are 10-1000 times that of the material costs. This cost discrepancy is attributed to the labor costs of manufacturing the column, as well as its quality control testing. Even with all the time consuming quality control testing, commercially available columns still suffer from packing inconsistencies in the column bed. Evidence of column-to-column variation is observed by the fact that brand new commercial columns of the same type, produce different backpressures when installed. Furthermore, the documentation shipped with columns often make the user aware that split ratios will need to be adjusted for each individual column. Split ratio adjustment between columns is necessary due to varying backpressures caused by inconsistent packing of the column beds. An example of a packed bed defect in a commercial small bore LC column (75 μm, 3 μm particles) is shown in FIG. 1. The polyimide-coated capillary 13 contains a packed bed 12. The large void 11 in the packed bed 12 is clearly visible. The void 11 creates a detrimental effect on the column's performance. Since the columns are manufactured in a non-automated manner, there is a great variation between columns of the same type, as the numbers and sizes of the voids greatly vary throughout the columns' packed beds. This variation results in performance variation even between columns of the same type.
An automated method for both manufacturing and quality control testing small bore LC columns is desired. An automated method allows for improved column-to-column reproducibility, and lower column costs. This improved reproducibility is imperative for nanoLC to enter into the field of clinical diagnostics.